Moesin fragments associated with immune thrombocytopenia

ABSTRACT

The present application provides compositions and methods useful for detecting and monitoring immune thrombocytopenia.

TECHNICAL FIELD

The present application relates to the field of molecular biology andmedical study with respect to autoimmune diseases. More specifically,the present application concerns methods and compositions based onunique presence of specific autoantibodies associated with immunethrombocytopenia.

BACKGROUND

Autoimmune diseases are diseases arising from aberrant response of theimmune system against one's own substances and tissues. There are morethan 80 different types of autoimmune diseases that, collectively,amount to the number two cause of chronic illness, and one of the top 10leading causes of death in women of all age groups up to 64 years.

Significant medical research efforts have been devoted to understandingthe mechanism of autoimmune diseases and finding effective diagnosis andtreatments therefore. Many autoimmune diseases are now characterized bythe presence and undesirable activities of autoantibodies. Theseautoantibodies recognize and bind to often normal and healthy selfantigens, thereby causing significant damages and failures of relevanttissues and organs.

Immune thrombocytopenia is an autoimmune hematological disease that ischaracterized by an attack by the immune system that destroys plateletsin the blood, resulting in an abnormally low platelet count. Theplatelet destruction is due to the presence of antiplateletautoantibodies, which are antibodies directed against the patient's ownplatelets. This low platelet count can lead to easy bruising, bleedinggums or nose and, less commonly, to severe internal bleeding.

In clinical practice, immune thrombocytopenia is difficult to diagnosedue to similarity in clinical symptoms with other diseases such asanaphylactoid purpura, myelodysplastic syndrome, multiple myeloma andother non-immune mediated thrombocytopenia. Generally, diagnosis ofimmune thrombocytopenia is a process of exclusion, and many clinicaltests need to be performed for exclusion of many other diseases (such asthe diseases described above) before reaching the diagnosis of immunethrombocytopenia. These clinical tests may take several months or evenlonger to go through and thus significantly delay proper treatment tothe patients.

Furthermore, some diseases such as aplastic anemia, acute leukemia,systemic lupus erythematosus, Wiskott-Aldrich syndrome, Evans syndromecan also cause immune thrombocytopenia in the course of diseasedevelopment and progression. Such immune thrombocytopenia is calledsecondary immune thrombocytopenia, to distinguish it from primary immunethrombocytopenia, which is caused originally by an autoimmune responseagainst the platelets. It is also importance to determine the onset ofsecondary immune thrombocytopenia in patients for timely treatment.

Therefore, one of the challenges in clinical treatment of immunethrombocytopenia is the accurate and early diagnosis of the disease in apatient.

DISCLOSURE OF THE INVENTION

The present application provides compositions and methods for diagnosingand monitoring immune thrombocytopenia based at least in part on thegeneration of moesin fragments from particular moesin functional domainsof the human moesin protein and their uses for detecting specificanti-moesin autoantibodies, whose presence and quantity in turncorrelate with diagnosis and prognosis of immune thrombocytopenia inpatients. Certain relevant terms used below in this section are definedin the Definitions section of this application.

In one aspect, the present application provides a method for diagnosingimmune thrombocytopenia comprising (i) contacting a sample from asubject suspected of immune thrombocytopenia with a first peptidecomprising a moesin fragment capable of binding to an anti-moesinautoantibody, wherein the moesin fragment consists essentially of theC-terminal tail domain of human moesin protein or a fragment thereof;(ii) detecting the binding of said first peptide to an anti-moesinautoantibody. Presence of the anti-moesin autoantibody binding to thefirst peptide in the sample at a level higher than the normal levelobtained from a reference sample is indicative of immunethrombocytopenia in the subject. Different levels of the anti-moesinautoantibody may be correlated with different stages and degrees ofseverity of immune thrombocytopenia in the subject.

In certain embodiments, the first peptide comprises at least eightconsecutive amino acid residues of the C-terminal tail domain of humanmoesin protein. In certain embodiments, the first peptide comprises atleast 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 consecutive amino acidresidues of the C-terminal tail domain of human moesin protein. Incertain embodiments, the first peptide comprises at least 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30consecutive amino acid residues of the C-terminal tail domain of humanmoesin protein.

In certain embodiments, the C-terminal tail domain of human moesinprotein consists of amino acid residues from the region between aboutamino acid residues 471-577 of the human moesin protein. In certainembodiments, the C-terminal tail domain of human moesin protein containsamino acid residues from the region between amino acid residues 471-574,471-575, 471-576, 471-577, 472-574, 472-575, 472-576, 472-577, 473-574,473-575, 473-576, 473-577, 474-574, 474-575, 474-576, or 474-577 of thehuman moesin protein. In certain embodiments, the C-terminal tail domainof human moesin protein contains amino acid residues selected from thegroup consisting of amino acid residues from the region between aminoacid residues 471-487, 488-501, 502-577, and 471-577 of human moesinprotein. In certain embodiments, the first peptide comprises the entireC-terminal tail domain of human moesin protein. In certain embodiments,the first peptide consists essentially of amino acid residues 471-577 ofthe human moesin protein or a fragment thereof. In certain embodiments,the first peptide does not contain any substantial portion of theN-terminal FERM domain of human moesin protein. As used herein, the term“substantial portion” refers to a portion of the relevant domain(Helical domain or N-terminal FERM domain or C-terminal tail domain)that can compete with such domain (Helical domain or N-terminal FERMdomain or C-terminal tail domain) for specific binding to an antibodycapable of binding to the entire relevant domain (Helical domain orN-terminal FERM domain or C-terminal tail domain).

In certain embodiments, the first peptide comprises at least eightconsecutive amino acid residues from the region between amino acidresidues 471-487 of the human moesin protein. In certain embodiments,the first peptide comprises at least eight consecutive amino acidresidues from the region between amino acid residues 488-501 of thehuman moesin protein. In certain embodiments, the first peptidecomprises at least eight consecutive amino acid residues from the regionbetween amino acid residues 502-577 of the human moesin protein.

In certain embodiments, the first peptide shares at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity withthe C-terminal tail domain of human moesin protein or a fragmentthereof. In certain embodiments, the first peptide shares at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequenceidentity with one of the amino acid sequences selected from the groupconsisting of amino acid residues 471-487, 488-501, 502-577, and 471-577of human moesin protein.

In another aspect, the present application provides a method fordiagnosing immune thrombocytopenia further comprising contacting asample from a subject suspected of immune thrombocytopenia with a secondpeptide capable of binding to an anti-moesin autoantibody, wherein thesecond peptide comprises a second moesin fragment consisting essentiallyof the N-terminal FERM domain of human moesin protein or a fragmentthereof; and detecting the binding of the second peptide to theanti-moesin autoantibodies. Presence of the anti-moesin autoantibodybinding to the second peptide at a level higher than a normal levelobtained from a reference sample is indicative of immunethrombocytopenia in the subject.

In certain embodiments, the second peptide comprises at least eightconsecutive amino acid residues of the N-terminal FERM domain of humanmoesin protein. In certain embodiments, the second peptide comprises atleast 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 consecutive amino acidresidues of the N-terminal FERM domain of human moesin protein. Incertain embodiments, the second peptide comprises at least 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or 30 consecutive amino acid residues of the N-terminal FERM domain ofhuman moesin protein.

In certain embodiments, the N-terminal FERM domain of human moesinprotein consists of amino acid residues from the region between aboutamino acid residues 1-297 of the human moesin protein. In certainembodiments, the N-terminal FERM domain of human moesin protein containsamino acid residues from the region between amino acid residues 1-294,1-295, 1-296, 1-297, 2-294, 2-295, 2-296, 2-297, 3-294, 3-295, 3-296,3-297, 4-294, 4-295, 4-296 or 4-297 of the human moesin protein. Incertain embodiments, the second peptide comprises the entire N-terminalFERM domain of human moesin protein. In certain embodiments, the secondpeptide consists essentially of amino acid residues of the N-terminalFERM domain of the human moesin protein or a fragment thereof. Incertain embodiments, the N-terminal FERM domain of human moesin proteincontains amino acid residues selected from the group consisting of aminoacid residues from the region between amino acid residues 1-94, 95-201,202-297, and 1-297 of human moesin protein. In certain embodiments, thesecond peptide does not contain any substantial portion of theC-terminal tail domain of human moesin protein.

In certain embodiments, the second peptide comprises at least eightconsecutive amino acid residues from the region between amino acidresidues 1-94 of the human moesin protein. In certain embodiments, thesecond peptide comprises at least eight consecutive amino acid residuesfrom the region between amino acid residues 95-201 of the human moesinprotein. In certain embodiments, the second peptide comprises at leasteight consecutive amino acid residues from the region between amino acidresidues 202-297 of the human moesin protein.

In certain embodiments, the second peptide shares at least 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identitywith the N-terminal FERM domain of human moesin protein or a fragmentthereof. In certain embodiments, the second peptide shares at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequenceidentity with one of the amino acid sequences selected from the groupconsisting of amino acid residues 1-94, 95-201, 202-297, and 1-297 ofhuman moesin protein.

In another aspect, the first and/or the second peptide described in thepresent application further comprises a carrier polypeptide. The term“carrier polypeptide” refers to any peptide or polypeptide that can beconjugated to the moesin fragment of the peptide of the presentapplication. A carrier polypeptide can be beneficial to the peptide ofthe present application, e.g. to promote the stability, solubility,specific or non-specific binding affinity and/or function of the peptideof the present application. However, a carrier polypeptide is notrequired to provide any benefit or even biological function to thepeptide of the present application. Commonly used carrier polypeptidesinclude human serum albumin, bovine serum albumin, antibody fragmentssuch as the antibody constant region.

In another aspect, the present application provides a method fordiagnosing immune thrombocytopenia, comprising contacting a sample froma subject suspected of immune thrombocytopenia with a first and secondpeptides capable of binding to anti-moesin autoantibodies, wherein thefirst peptide comprises a first moesin fragment consisting essentiallyof the C-terminal tail domain of human moesin protein or a fragmentthereof, the second peptide comprises a second moesin fragmentconsisting essentially of the N-terminal FERM domain of human moesinprotein or a fragment thereof, and detecting the binding of the firstand second peptide to the anti-moesin autoantibodies. Presence of theanti-moesin autoantibodies binding to the first and second peptides atlevels higher than a normal level obtained from a reference sample isindicative of immune thrombocytopenia in the subject. The differentlevels of the anti-moesin autoantibodies binding to the first and secondpeptides, respectively, may be correlated with the different stages anddegrees of severity of immune thrombocytopenia in a subject. In certainembodiments, the sample is tested for binding of the second peptide tothe anti-moesin antibodies before tested for binding of the firstpeptide to the anti-moesin antibodies. In certain embodiments, thesample is tested for binding of the first and second peptides to theanti-moesin antibodies at the same time. In certain embodiments, thesample is tested for binding of the second peptide to the anti-moesinantibodies after tested for binding of the first peptide to theanti-moesin antibodies, and then tested at higher concentration of thesample for binding of the first peptide to the anti-moesin antibodiesagain.

In another aspect, the present application provides the use of a peptidecapable of binding to an anti-moesin autoantibody in the manufacture ofa diagnostic composition for the diagnosis of immune thrombocytopenia ina subject, wherein the peptide consists essentially of the C-terminaltail domain of human moesin protein or a fragment thereof.

In another aspect, the present application provides the use of a peptidecapable of binding to an anti-moesin autoantibody in the manufacture ofa diagnostic composition for the diagnosis of immune thrombocytopenia ina subject, wherein the peptide consists essentially of the C-terminaltail domain of human moesin protein or a fragment thereof, and thepeptide comprises at least eight consecutive amino acid residues of theC-terminal tail domain of human moesin protein.

In another aspect, the present application provides the use of first andsecond peptides capable of binding to anti-moesin autoantibodies in themanufacture of a diagnostic composition for the diagnosis of immunethrombocytopenia in a subject, wherein the first peptide consistsessentially of the C-terminal tail domain of human moesin protein or afragment thereof, and the second peptide consists essentially of theN-terminal FERM domain of human moesin protein or a fragment thereof.

In another aspect, the present application provides a kit for diagnosingimmune thrombocytopenia, comprising a peptide capable of binding to ananti-moesin autoantibody, wherein the peptide comprises a moesinfragment consisting essentially of the C-terminal tail domain of humanmoesin protein or a fragment thereof, and a detecting reagent. Incertain embodiments, the detecting reagent is an antibody capable ofbinding to the anti-moesin autoantibody. In certain embodiments, thepeptide capable of binding to an anti-moesin autoantibody is bound to asolid phase.

In another aspect, the present application provides a kit for diagnosingimmune thrombocytopenia, comprising a first peptide capable of bindingto an anti-moesin autoantibody, wherein the first peptide comprises afirst moesin fragment consisting essentially of the C-terminal taildomain of human moesin protein or a fragment thereof, a second peptidecapable of binding to an anti-moesin autoantibody, wherein the secondpeptide comprises a second moesin fragment consisting essentially of theN-terminal FERM domain of human moesin protein or a fragment thereof,and a detecting reagent.

In another aspect, the present application provides a method ofdetermining the pathological state of a subject having immunethrombocytopenia, comprising the following steps:

-   -   (i) contacting a sample from a subject suspected of having        immune thrombocytopenia with a composition comprising a peptide        capable of binding to an anti-moesin autoantibody, wherein the        peptide comprises a moesin fragment consisting essentially of        the C-terminal tail domain of human moesin protein or a fragment        thereof;    -   (ii) detecting the binding of the peptide to an anti-moesin        autoantibody and measuring the level of the anti-moesin        autoantibody bound to the peptide; and    -   (iii) determining the pathological state of the subject        according to a comparison of the level of the anti-moesin        autoantibody to a reference database obtained from diseased        reference samples correlating titers of the anti-moesin        autoantibody to pathological states of the immune        thrombocytopenia.

In certain embodiments, the reference database is a reference curvewhich shows the relationship between the titers of the anti-moesinautoantibodies and the levels of platelet counts in the subject.

In another aspect, the present application provides a method ofmonitoring treatment response in a subject undergoing a treatment forimmune thrombocytopenia, comprising:

-   -   (i) contacting a sample from a subject suspected of having        immune thrombocytopenia with a peptide capable of binding to an        anti-moesin autoantibody, wherein the peptide comprises a moesin        fragment consisting essentially of the C-terminal tail domain of        human moesin protein or a fragment thereof;    -   (ii) detecting the binding of said peptide to an anti-moesin        autoantibody and measuring the level of the anti-moesin        autoantibody bound to the peptide; and    -   (iii) determining the pathological state of the subject        according to a comparison of the level of the anti-moesin        autoantibody to a reference database obtained from diseased        reference samples correlating titers of the anti-moesin        autoantibody to pathological states of the immune        thrombocytopenia, wherein a decrease in titer is indicative of        positive response of the subject to the treatment.

In certain embodiments, the reference database contains data for thelevels of the anti-moesin autoantibodies at different stages of thetreatment.

In another aspect, the application provides a method of diagnosing animmune thrombocytopenia in a subject, comprising the following steps:(i) contacting a peptide comprising at least eight consecutive aminoacid residues of the C-terminal tail domain of human moesin protein witha sample obtained from said subject; and (ii) determining whether theanti-moesin autoantibody is present in said sample at a level greaterthan the level of said anti-moesin autoantibody in a reference sample,thereby indicating that the subject has immune thrombocytopenia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Amino acid sequence of the full length human moesin protein (SEQID NO:1, also referred to herein as Moesin-5).

FIG. 2. Amino acid sequence of moesin fragments: Moesin-1 (SEQ ID NO:2),Moesin-2 (SEQ ID NO:3), Moesin-3 (SEQ ID NO:4) and Moesin-4 (SEQ IDNO:5).

FIG. 3. cDNA sequence encoding for the full length human moesin protein(SEQ ID NO:6).

FIG. 4. Cloning map of the pET32a(+) expression vector.

FIG. 5. Cloning map of the pET28a(+) expression vector.

FIG. 6 Graph illustrating the titers of anti-autoantibody to specificmoesin fragments in sera of different patient groups.

MODES FOR CARRYING OUT THE INVENTION

The practice of the present application will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” series(Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M.Ausubel et al., eds., 1987, and periodic updates); “PCR: The PolymeraseChain Reaction”, (Mullis et al., eds., 1994). Primers, polynucleotidesand polypeptides employed in the present application can be generatedusing standard techniques known in the art.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al., Dictionary ofMicrobiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanismsand Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992), provideone skilled in the art with a general guide to many of the terms used inthe present application.

DEFINITIONS

The term “moesin” stands for membrane-organizing extension spikeprotein, as described in Lankes and Furthmayr (1991) Proc. Natl. Acad.Sci., 88:8297-8301. Full length human moesin protein is a 577 amino acidpolypeptide having an amino acid sequence as set forth in FIG. 1 (SEQ IDNO:1). The moesin protein consists of three domains: the N-terminal FERMdomain, the helical domain, and the C-terminal tail domain, as furtherdefined below. It belongs to the ERM (ezrin-radixin-moesin) family. Thethree ERM proteins, primarily expressed in cytoplasm right beneath theplasma membrane, share high degrees of sequence homology and act aslinking proteins between the plasma membrane and the actin cytoskeleton.Furthermore, human moesin protein shares high degrees of sequencehomology with moesins from other species such as mouse and bovinemoesins. Sato et al. (1992) J. Cell Sci. 103:131-143.

The term “moesin fragment” refers to a portion of the moesin polypeptidethat is shorter than the full length wild type moesin protein, and thatis capable of binding to an anti-moesin autoantibody. Useful in thepresent application are such moesin fragments capable of binding todomain-specific anti-moesin autoantibodies. A “fragment” of the moesinfragment means a portion of the moesin fragment that is shorter thansuch moesin fragment, and that retains the ability of binding to ananti-moesin autoantibody.

The “N-terminal FERM domain” of human moesin protein refers to theglobular portion of the wild type human moesin protein structurallyproximate to the amino-terminal of the protein and functionallyresponsible for localizing the protein to the plasma membrane andinteracting with adhesion molecules. The FERM domain, which stands forband four-point-one, ezrin, radixin, moesin homology domain because ofits homology with the band 4.1 protein, defines members of the band 4.1superfamily, which includes cytoskeletal proteins such as erythrocyteband 4.1, talin, and the ezrin-radixin-moesin (ERM) protein family, aswell as several tyrosine kinases and phosphatases and the tumorsuppressor protein merlin. Specifically, the term refers to the firstabout 297 amino acid residues of the mature form of human moesin protein(e.g., amino acid residues 1-297 (SEQ ID NO:2)). In certain literatures,the same domain is also known as N-ERM associated domain (N-ERMAD),which is included in the definition herein. Bretscher et al. (1995)Biochem. 34, 16830-7.

The “C-terminal tail domain” of human moesin protein refers to theportion of the wild type human moesin protein structurally proximate tothe carboxy-terminal of the protein and functionally responsible forbinding to and interacting with actin filaments. The tail domain ofmoesin is positively charged and adopts an extended, meanderingstructure. Specifically, the term refers to the last about 107 aminoacid residues of human moesin protein (e.g., amino acid residues 471-577(SEQ ID NO:5)). In certain literatures, the same domain is also known asC-ERM associated domain (C-ERMAD), which is included in the definitionherein. Bretscher et al. (1995). The last 34 amino acid residues of theC-terminal tail domain are highly conserved amongst ERM proteins andforms the region for binding to F-actin. Within the F-actin bindingregion, there exists a threonine residue (Thr558 in wild type humanmoesin) that is phosphorylated during the activation of the protein.

The “helical domain” of human moesin protein refers to the centralportion of the wild type human moesin resided in between the N-terminalFERM domain and the C-terminal tail domain. It adopts an extendedalpha-helical structure, acting as a linker between the two terminaldomains. Specifically, the term refers to the region encompassing aboutamino acid residues 298-470 of human moesin protein (SEQ ID NO:4).

The term “anti-moesin autoantibody” refers to an anti-moesin antibodyproduced by an individual's immune system that recognizes and binds tosuch individual's own moesin protein or fragments thereof. The presenceof anti-moesin autoantibody can be associated with an immunethrombocytopenia, and the titer of such anti-moesin autoantibody in thebody may correlate to the pathological state of the immunethrombocytopenia.

The term “diagnosis” is used herein to refer to the identification of amolecular or pathological state, disease or condition, such as theidentification of an autoimmune disease, or to refer to identificationof a patient with autoimmune disease who may benefit from a particulartreatment regimen. In one embodiment, diagnosis refers to theidentification of immune thrombocytopenia. In yet another embodiment,diagnosis refers to the identification of immune thrombocytopeniaassociated with higher than normal presence of anti-moesinautoantibodies in a subject.

The term “prognosis” is used herein to refer to the prediction of thelikelihood of outcomes of disease symptoms, including, for example,recurrence, flaring, and drug resistance, of a disease. The term alsorefers to the prediction of the likelihood of clinical benefit from atherapy.

The term “prediction” is used herein to refer to the likelihood that apatient will respond either favorably or unfavorably to a drug or set ofdrugs or a particular therapy course. In one embodiment, the predictionrelates to the extent of those responses. In one embodiment, theprediction relates to whether and/or the probability that a patient willsurvive or improve following treatment, for example treatment with aparticular therapeutic agent, and for a certain period of time withoutdisease recurrence. The predictive methods of the invention can be usedclinically to make treatment decisions by choosing the most appropriatetreatment modalities for any particular patient. The predictive methodsof the present application are valuable tools in predicting if a patientis likely to respond favorably to a treatment regimen, such as a giventherapeutic regimen, including for example, administration of a giventherapeutic agent or combination, surgical intervention, steroidtreatment, etc., or whether long-term survival of the patient, followinga therapeutic regimen is likely.

The term “thrombocytopenia” is used herein to refer to any disorder inwhich the platelet level in a subject fall below a normal range ofplatelet numbers for that individual, due to disturbance in theproduction or destruction of platelet. In one embodiment, normal bloodplatelet levels range from about 150.000 to 300.000 per microliterperipheral blood in humans. Thrombocytopenia as used herein also refersto a decrease in platelet number in an individual when compared to theplatelet number measured at a certain reference point in thatindividual. The reference point mentioned can be, for instance, thestart of a therapy such as a radiation therapy or chemotherapy.

The term “immune thrombocytopenia” is used herein to refer to any typeof thrombocytopenia arising from an auto-immune response directedagainst an individual's own platelets. Immune thrombocytopenia includesprimary immune thrombocytopenia, in which autoimmune response is theoriginal cause for the decrease in the platelet counts. Immunethrombocytopenia includes, for example, idiopathic thrombocytopenicpurpura. Furthermore, there is secondary immune thrombocytopenia, inwhich the decrease in platelet counts is associated with one or moreother diseases such as aplastic anemia, iron deficiency anemia andautoimmune hemolytic anemia, leukemia, systemic lupus erythematosus,HIV-associated thrombocytopenia, Wiskott-Aldrich syndrome, Evanssyndrome and the like. In secondary immune thrombocytopenia, those otherdiseases induce or trigger or otherwise cause an individual's body togenerate an auto-immune response against its own platelets.

“Sample” or “test sample” herein refers to a composition that isobtained or derived from a subject of interest that contains a cellularand/or other molecular entity that is to be characterized and/oridentified, for example based on physical, biochemical, chemical and/orphysiological characteristics. In one embodiment, the definitionencompasses blood and other liquid samples of biological origin andtissue samples such as a biopsy specimen or tissue cultures or cellsderived there from or cell cultures. The source of the tissue sample maybe solid tissue as from a fresh, frozen and/or preserved organ or tissuesample or biopsy or aspirate; blood or any blood constituents such asplasma or serum; bodily fluids; and cells from any time in gestation ordevelopment of the subject or plasma. In another embodiment, the sampleis whole blood, serum or plasma obtained from a subject. A subject canbe a human or an animal subject. In another embodiment, a subject has oris suspected of having an immune thrombocytopenia. In anotherembodiment, the definition includes biological samples that have beenmanipulated in any way after their procurement, such as by treatmentwith reagents, solubilization, or enrichment for certain components,such as proteins or polynucleotides.

In one embodiment, a sample is obtained from a subject or patient priorto any treatment. In another embodiment, a test sample is obtainedduring or after treatment such as immune thrombocytopenia therapy. Inone embodiment, the test sample is a clinical sample. In anotherembodiment, the test sample is used in a diagnostic assay. In anotherembodiment, the sample is pre-tested with other known blood testingmethods before being tested with the methods of the present application.These blood testing methods include, for example, full blood count,liver enzymes, renal function, vitamin B₁₂ levels, folic acid levels,erythrocyte sedimentation rate, peripheral blood smear, bone marrowbiopsy and the like.

A “reference sample”, as used herein, refers to a sample from a sourceknown, or believed, not to be afflicted with the disease or conditionfor which a method or composition of the present application is beingused to identify. In one embodiment, a reference sample is obtained froma healthy part of the body of the same subject or patient in whom adisease or condition is being identified using a composition or methodof the present application. In one embodiment, a reference sample isobtained from a healthy part of the body of an individual who is not thesubject or patient in whom a disease or condition is being identifiedusing a composition or method of the present application. In oneembodiment, the reference sample is a sample from a healthy individualthat has a normal platelet count.

A “disease reference sample”, as used herein, refers to a sample from asource that is clinically identified as being afflicted with the diseaseor condition for which a method or composition of the presentapplication is being used to identify. In one embodiment, the diseasereference sample is a sample obtained from a subject or patient that hasbeen clinically diagnosed with immune thrombocytopenia. In oneembodiment, the subject or patient that has been clinically diagnosedwith immune thrombocytopenia is under treatment for immunethrombocytopenia.

A “reference database”, as used herein, refers to a collection of data,standard, or level from one or more reference samples or diseasereference samples. In one embodiment, such collection of data, standardor level are normalized so that they can be used for comparison purposewith data from one or more sample. “Normalize” or “normalization” is aprocess by which a measurement raw data is converted into data that maybe directly compared with other so normalized data. Normalization isused to overcome assay-specific errors caused by factors that may varyfrom one assay to another, for example, variation in loaded quantities,binding efficiency, detection sensitivity, and other various errors. Inone embodiment, a reference database includes titers of anti-moesinautoantibodies, platelet counts, blood cell counts, and/or otherlaboratory and clinical data from one or more reference samples ordisease reference samples. In one embodiment, a reference databaseincludes levels of anti-moesin autoantibodies that are each normalizedas a percent of the level of anti-moesin autoantibody of a controlsample (e.g. a known amount of anti-moesin autoantibody) tested underthe same conditions as the reference samples or disease referencesamples. In order to compare with such normalized levels of anti-moesinautoantibodies, the level of anti-moesin autoantibody of a test sampleis also measured and calculated as a percent of the level of anti-moesinautoantibody of a control sample tested under the same conditions as thetest sample. In one embodiment, a reference database is established bycompiling reference sample data from healthy subjects and/ornon-diseased part of the body of the same subject or patient in whom adisease or condition is being identified using a composition or methodof the present application. In one embodiment, a reference database isestablished by compiling data from disease reference samples fromindividuals under treatment for immune thrombocytopenia. In oneembodiment, a reference database is established by compiling data fromdisease reference samples from individuals at different stages of immunethrombocytopenia as evidenced by, for example, different levels ofplatelet counts and other clinical indications.

In certain embodiments, the term “increase” refers to an overallincrease of 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or greater, in the level of autoantibody,detected by standard art known methods such as those described herein,as compared to a reference sample or a disease reference sample. Incertain embodiments, the term increase refers to the increase in thelevel of autoantibody in the sample wherein the increase is at leastabout 1.25×, 1.5×, 1.75×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 25×, 50×,75×, or 100× the level of the autoantibody in the reference sample orthe disease reference sample.

In certain embodiments, the term “decrease” herein refers to an overallreduction of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in thelevel of autoantibody, detected by standard art known methods such asthose described herein, as compared to a reference sample or a diseasereference sample. In certain embodiments, the term decrease refers tothe decrease in the level of autoantibody in the sample wherein thedecrease is at least about 0.9×, 0.8×, 0.7×, 0.6×, 0.5×, 0.4×, 0.3×,0.2×, 0.1×, 0.05×, or 0.01× the level of autoantibody in the referencesample or the disease reference sample.

The term “detection means” refers to a moiety or technique used todetect the presence of the detectable antibody in the ELISA herein andincludes detection agents that amplify the immobilized label such aslabel captured onto a microtiter plate. In one embodiment, the detectionmeans is a colorimetric detection agent such as avidin orstreptavidin-HRP. In another embodiment, the detection means is aH₂O₂/TMB coloring system.

The term “capture reagent” refers to a reagent capable of binding andcapturing a target molecule in a sample such that under suitablecondition, the capture reagent-target molecule complex can be separatedfrom the rest of the sample. Typically, the capture reagent isimmobilized or immobilizable. In a sandwich immunoassay, the capturereagent is preferably an antibody or a mixture of different antibodiesagainst a target antigen.

By “correlate” or “correlating” is meant comparing, in any way, theperformance and/or results of a first analysis or protocol with theperformance and/or results of a second analysis or protocol. Forexample, one may use the results of a first analysis or protocol incarrying out a second protocols and/or one may use the results of afirst analysis or protocol to determine whether a second analysis orprotocol should be performed. With respect to the embodiment ofautoantibody detection, one may use the results of the detectionanalysis or protocol to determine whether a specific therapeutic regimenshould be performed.

The word “label” when used herein refers to a compound or compositionwhich is conjugated or fused directly or indirectly to a reagent such asa nucleic acid probe or an antibody and facilitates detection of thereagent to which it is conjugated or fused. The label may itself bedetectable (e.g., radioisotope labels or fluorescent labels) or, in thecase of an enzymatic label, may catalyze chemical alteration of asubstrate compound or composition which is detectable.

An “isolated” polypeptide is one that has been identified and separatedand/or recovered from contaminant components of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In certain embodiments, the polypeptide willbe purified (1) to greater than 95% by weight of polypeptide asdetermined by the Lowry method, or more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue, or silver stain. Isolated polypeptide includes thepolypeptide in situ within recombinant cells since at least onecontaminant component of the polypeptide's natural environment will notbe present. Ordinarily, however, isolated polypeptide will be preparedby at least one purification step.

“Percent (%) amino acid sequence identity” with respect to a moesindomain or fragment of the present application is defined as thepercentage of amino acid residues in a sequence of interest that areidentical with the amino acid residues in the moesin domain or fragment,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity, and not considering anyconservative amino acid substitutions as part of the sequence identity.Alignment for purposes of determining percent amino acid sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. See, forexample, Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997);Altschul et al., Methods in Enzymology 266:460-480 (1996). Those skilledin the art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull length of the sequences being compared.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length or intact monoclonalantibodies), polyclonal antibodies, multivalent antibodies,multispecific antibodies (e.g., bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired antigen binding activity.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

Responsiveness of a patient can be assessed using any endpointindicating a benefit to the patient, including, without limitation, (1)inhibition, to some extent, of disease progression, including slowingdown and complete arrest; (2) reduction in the number of diseaseepisodes and/or symptoms; (3) reduction in lesion size; (4) inhibition(i.e., reduction, slowing down or complete stopping) of disease cellinfiltration into adjacent peripheral organs and/or tissues; (5)inhibition (i.e. reduction, slowing down or complete stopping) ofdisease spread; (6) relief, to some extent, of one or more symptomsassociated with the disorder; (7) increase in the length of disease-freepresentation following treatment; (8) decrease of auto-immune response,which may, but does not have to, result in the regression or ablation ofthe disease lesion, e.g., progression-free survival; (9) increasedoverall survival; (10) higher response rate; and/or (11) decreasedmortality at a given point of time following treatment. The term“benefit” is used in the broadest sense and refers to any desirableeffect.

Typical Methods and Materials of the Invention

The present application provides compositions and methods for diagnosingand monitoring immune thrombocytopenia associated with the presence andtiter of anti-moesin autoantibodies. Conventional methods known to theskilled in the art can be used to carry out the present application.

Vectors, Host Cells and Recombinant Methods

The polypeptides of the present application can be producedrecombinantly, using techniques and materials readily obtainable. Forrecombinant production of a polypeptide of the present application, thenucleic acid encoding it is isolated and inserted into a replicablevector for further cloning (amplification of the DNA) or for expression.DNA encoding the polypeptide of the present application is readilyisolated and sequenced using conventional procedures. For example, a DNAencoding a human moesin protein is isolated and sequenced, e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the protein. Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moreselection genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

Signal Sequence Component

Polypeptides of the present application may be produced recombinantlynot only directly, but also as a fusion polypeptide with a heterologouspolypeptide, which is typically a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected typically isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells, the signalsequence can be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, 1 pp, orheat-stable enterotoxin II leaders. For yeast secretion, the signalsequence may be, e.g., the yeast invertase leader, α factor leader(including Saccharomyces and Kluyveromyces α-factor leaders), or acidphosphatase leader, the C. albicans glucoamylase leader, or the signaldescribed in WO 90/13646. In mammalian cell expression, mammalian signalsequences as well as viral secretory leaders, for example, the herpessimplex gD signal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the polypeptide of the present application.

Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take upnucleic acid, such as DHFR, thymidine kinase, metallothionein-I and -II,typically primate metallothionein genes, adenosine deaminase, ornithinedecarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding a polypeptide of the present application, wild-type DHFRprotein, and another selectable marker such as aminoglycoside3′-phosphotransferase (APH) can be selected by cell growth in mediumcontaining a selection agent for the selectable marker such as anaminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S.Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid Yrp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

Promotor Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to a nucleic acidencoding a polypeptide of the present application. Promoters suitablefor use with prokaryotic hosts include the phoA promoter, β-lactamaseand lactose promoter systems, alkaline phosphatase, a tryptophan (trp)promoter system, and hybrid promoters such as the tac promoter. However,other known bacterial promoters are suitable. Promoters for use inbacterial systems also will contain a Shine-Dalgarno (S.D.) sequenceoperably linked to the DNA encoding the polypeptide of the presentapplication.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldyhyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Transcription of polypeptides of the present application from vectors inmammalian host cells is controlled, for example, by promoters obtainedfrom the genomes of viruses such as polyoma virus, fowlpox virus,adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the rous sarcoma virus long terminal repeat can be used as the promoter.

Enhancer Element Component

Transcription of a DNA encoding a polypeptide of this application byhigher eukaryotes is often increased by inserting an enhancer sequenceinto the vector. Many enhancer sequences are now known from mammaliangenes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, one will use an enhancer from a eukaryotic cell virus.Examples include the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) onenhancing elements for activation of eukaryotic promoters. The enhancermay be spliced into the vector at a position 5′ or 3′ to thepolypeptide-encoding sequence, but is typically located at a site 5′from the promoter.

Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the polypeptide of the present application.One useful transcription termination component is the bovine growthhormone polyadenylation region. See WO94/11026 and the expression vectordisclosed therein.

Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing DNA encoding thepolypeptides of the present application in the vectors herein are theprokaryote, yeast, or higher eukaryote cells described above. Suitableprokaryotes for this purpose include eubacteria, such as Gram-negativeor Gram-positive organisms, for example, Enterobacteriaceae such asEscherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus,Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratiamarcescans, and Shigella, as well as Bacilli such as B. subtilis and B.licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710published 12 Apr. 1989), Pseudomonas such as P. aeruginosa andStreptomyces. Typically, the E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coli B, E. coli BL21(DE3), E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for vectorsencoding polypeptide of the present application. Saccharomycescerevisiae, or common baker's yeast, is the most commonly used amonglower eukaryotic host microorganisms. However, a number of other genera,species, and strains are commonly available and useful herein, such asSchizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis,K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii(ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906),K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichiapastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234);Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis;and filamentous fungi such as, e.g., Neurospora, Penicillium,Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of polypeptides of the presentapplication can be derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent application, particularly for transfection of Spodopterafrugiperda cells. Plant cell cultures of cotton, corn, potato, soybean,petunia, tomato, and tobacco can also be utilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for production of polypeptide of the present applicationand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Culturing the Host Cells

The host cells used to produce polypeptides of the present applicationmay be cultured in a variety of media. Commercially available media suchas Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma),RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM),Sigma) are suitable for culturing the host cells. In addition, any ofthe media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes etal., Anal. Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866;4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S.Pat. No. Re. 30,985 may be used as culture media for the host cells. Anyof these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN™drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

Chemical Synthesis of Peptides

The peptides of the present application can also be produced by chemicalsynthesis, for example, the solid phase synthesis method described byMerrifield in J.A.C.S. 85: 2149-2154 (1963) or the standard solutionsynthesis method described in “Peptide Synthesis” by Bodanszky, et al,second edition, John Wiley and Sons, 1976. These books are entirelyincorporated herein by reference.

The general procedure of the solid phase method of synthesis of apeptide involves initially attaching the protected C-terminal amino acidof the peptide to the resin. After attachment the resin is filtered,washed and the protecting group (e.g. t-butyloxycarbonyl) on the alphaamino group of the C-terminal amino acid is removed. The removal of thisprotecting group must take place, of course, without breaking the bondbetween that amino acid and the resin. To the resulting resin peptide isthen coupled the penultimate C-terminal protected amino acid. Thiscoupling takes place by the formation of an amide bond between the freecarboxy group of the second amino acid and the amino group of the firstamino acid attached to the resin. This sequence of events is repeatedwith successive amino acids until all amino acids of the peptide areattached to the resin. Finally, the protected peptide is cleaved fromthe resin and the protecting groups removed to obtain the desiredpeptide. The cleavage techniques used to separate the peptide from theresin and to remove the protecting groups depend upon the selection ofresin and protecting groups and are known to those familiar with the artof peptide synthesis.

The resin mentioned above may be any suitable polymer and shall containa functional group to which the first protected amino acid can be firmlylinked by a covalent bond. Various polymers are suitable for thispurpose, such as cellulose, polyvinyl alcohol, polymethylmethacrylate,and polystyrene. Appropriate protecting groups usable in solid phasesynthesis include t-butyloxycarbonyl (BOC), benzyl (BZL),t-amyloxycarbonyl (AOC), tosyl (TOS), o-bromophenylmethoxycarbonyl(BrZ), 2,6-dichlorobenzyl (BZLCl.sub.2), and phenylmethoxycarbonyl (Z orCBZ). Additional protecting groups are also described in J. F. W.McOmie, “Protective Groups in Organic Chemistry”, Plenum Press, NewYork, 1973. This book is entirely incorporated herein by reference.

The standard solution synthesis method can be performed by eitherstepwise or block coupling of amino acids or peptide fragments usingchemical or enzymatic methods of amide bond formation. These solutionsynthesis methods are well known in the art.

Polypeptide Purification

A polypeptide or protein of the present application may be recoveredfrom a subject. When using recombinant techniques, a polypeptide of thepresent application can be produced intracellularly, in the periplasmicspace, or directly secreted into the medium. Polypeptides of the presentapplication may be recovered from culture medium or from host celllysates. If membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of a polypeptide of the presentapplication can be disrupted by various physical or chemical means, suchas freeze-thaw cycling, sonication, mechanical disruption, or celllysing agents.

If a peptide is chemically synthesized, the peptide of the presentapplication may be recovered from the reaction medium by any suitabletechniques capable of separating the desired peptide from othercomponents in the medium. For a solid phase synthesis, the protectedpeptide is firstly cleaved off the resin using a suitable cleavingsolution. The selection of cleaving solution depends upon the propertiesof the resin and the amino acid bound thereto (such as trifluoroaceticacid for FMOC method). Cleaving is usually carried out under acidcondition. Upon completion of cleaving, a dissociative peptide is thenobtained and further purified using any suitable techniques (such as themethods described below).

The following procedures are exemplary of suitable protein purificationprocedures: by fractionation on an ion-exchange column; ethanolprecipitation; reverse phase HPLC; chromatography on silica,chromatography on heparin SEPHAROSE™, chromatography on an anion orcation exchange resin (such as a polyaspartic acid column, DEAE, etc.);chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; protein A Sepharosecolumns to remove contaminants such as IgG; and metal chelating columnsto bind epitope-tagged forms of polypeptides of the present application.Various methods of protein purification may be employed and such methodsare known in the art and described for example in Deutscher, Methods inEnzymology, 182 (1990); Scopes, Protein Purification: Principles andPractice, Springer-Verlag, New York (1982). The purification step(s)selected will depend, for example, on the nature of the productionprocess used and the particular polypeptide of the present applicationproduced.

Detection Methods

In the methods of the present application, a biological sample isobtained from a subject suspected of immune thrombocytopenia andexamined for expression of one or more anti-moesin autoantibodies.Expression of various anti-moesin autoantibodies in a sample can beanalyzed by a number of methodologies, many of which are known in theart and understood by the skilled artisan, including but not limited to,enzyme-linked immunosorbent assay (ELISA), enzyme-linked immuno-flowassay (ELIFA), immunoblotting, Western blot analysis,immunohistochemical analysis, immunoprecipitation, molecular bindingassays and the like. Multiplexed immunoassays such as those availablefrom Rules Based Medicine or Meso Scale Discovery (MSD) may also beused. These methods include both single-site and two-site or “sandwich”assays of the non-competitive types, as well as in the traditionalcompetitive binding assays. Detection can be conducted in vitro, in vivoor ex vivo.

Sandwich assays are among the most useful and commonly used assays. Anumber of variations of the sandwich assay technique exist, and all areintended to be encompassed by the present application. Briefly, in atypical forward sandwich assay, an unlabelled capture reagent (e.g., amoesin fragment) is immobilized on a solid substrate, and the sample tobe tested for the target protein (e.g., an anti-moesin autoantibody) isbrought into contact with the bound molecule. After a suitable period ofincubation, for a period of time sufficient to allow formation of anantibody-antigen complex, a detection antibody specific to the targetprotein (e.g., through binding to the Fc region of the anti-moesinautoantibody), labelled with a reporter molecule capable of producing adetectable signal is then added and incubated, allowing time sufficientfor the formation of another complex of capture reagent-targetprotein-detection antibody. Any unreacted material is washed away, andthe presence of the target protein is determined by observation of asignal produced by the reporter molecule. The results may either bequalitative, by simple observation of the visible signal, or may bequantitated by comparing with a control sample containing known amountsof the reporter molecules.

In a typical forward sandwich assay, a capture reagent havingspecificity for the target protein is either covalently or passivelybound to a solid support. The solid support is typically glass or apolymer, the most commonly used polymers being cellulose,polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.The solid supports may be in the form of tubes, beads, discs ofmicroplates, or any other surface suitable for conducting animmunoassay.

Variations on the forward assay include a simultaneous assay, in whichboth the sample and detection antibody are added simultaneously to thecapture reagent. These techniques are well known to those skilled in theart, including any minor variations as will be readily apparent. Anotheralternative method involves immobilizing the target proteins in thesample and then exposing the immobilized target proteins to the peptidesof the present application which may or may not be labeled with areporter molecule. Depending on the amount of target proteins and thestrength of the reporter molecule signal, a bound target protein may bedetectable by direct labeling with the capture reagent (e.g. a moesinfragment). Alternatively, a second detection antibody, specific to thecapture reagent is exposed to the target protein-capture reagent complexto form a target protein-capture reagent-detection antibody tertiarycomplex. The complex is detected by the signal emitted by the reportermolecule.

The term “reporter molecule”, as used herein, is meant a molecule which,by its chemical nature, provides an analytically identifiable signalwhich allows the detection of antigen-bound antibody. The most commonlyused reporter molecules in this type of assay are either enzymes,fluorophores or radionuclide containing molecules (i.e. radioisotopes)and chemiluminescent molecules.

In certain embodiments, the reporter molecules are enzymes conjugated tothe detection antibodies. The enzyme generally catalyzes a chemicalalteration of the chromogenic substrate that can be measured usingvarious techniques. For example, the enzyme may catalyze a color changein a substrate, which can be measured spectrophotometrically.Alternatively, the enzyme may alter the fluorescence orchemiluminescence of the substrate. When activated by illumination withlight of a particular wavelength, the fluorochrome adsorbs the lightenergy, inducing a state to excitability in the molecule, followed byemission of the light at a characteristic color visually detectable witha light microscope. The chemiluminescent substrate becomeselectronically excited by a chemical reaction and may then emit lightwhich can be measured (using a chemiluminometer, for example) or donatesenergy to a fluorescent acceptor. Examples of enzymatic labels includeluciferases (e.g., firefly luciferase and bacterial luciferase; U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malatedehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO),alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases (e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase), heterocyclic oxidases (such asuricase and xanthine oxidase), lactoperoxidase, microperoxidase, and thelike. Techniques for conjugating enzymes to antibodies are described inO'Sullivan et ah, Methods for the Preparation of Enzyme-AntibodyConjugates for use in Enzyme Immunoassay, in Methods in Enzym. (ed. J.Langone & H. Van Vunakis), Academic press, New York, 73:147-166 (1981).

Examples of enzyme-substrate combinations include, for example: (i)Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate,wherein the hydrogen peroxidase oxidizes a dye precursor (e.g.,orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB)); (ii) alkaline phosphatase (AP) withpara-Nitrophenyl phosphate as chromogenic substrate; and (iii)β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate (e.g.,4-methylumbelliferyl-β-D-galactosidase). Numerous other enzyme-substratecombinations are available to those skilled in the art. For a generalreview of these, see U.S. Pat. Nos. 4,275,149 and 4,318,980.

In certain embodiments, the reporter molecules are fluorophoresincluding, but are not limited to, rare earth chelates (europiumchelates), Texas Red, rhodamine, fluorescein, dansyl, Lissamine,umbelliferone, phycocrytherin, phycocyanin, or commercially availablefluorophores such SPECTRUM ORANGE7 and SPECTRUM GREEN7 and/orderivatives of any one or more of the above. The fluorophores can beconjugated to the antibody using the techniques disclosed in CurrentProtocols in Immunology, Volumes 1 and 2, Coligen et al, Ed.Wiley-Interscience, New York, Pubs. (1991), for example. Fluorescencecan be quantified using a fluorimeter.

In certain embodiments, the report molecules are radioisotopes, such as³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The detection antibody or capture reagentcan be labeled with the radioisotope using the techniques described inCurrent Protocols in Immunology, supra, for example and radioactivitycan be measured using scintillation counting.

Sometimes, the label is indirectly conjugated with the detectionantibody or capture reagent. The skilled artisan will be aware ofvarious techniques for achieving this. For example, the detectionantibody can be conjugated with biotin and the label can be conjugatedwith avidin, or vice versa. Biotin binds selectively to avidin and thus,the label can be conjugated with the detection antibody in this indirectmanner. Alternatively, to achieve indirect conjugation of the label withthe detection antibody, the detection antibody is conjugated with asmall hapten and the label is conjugated with an anti-hapten antibody.Thus, indirect conjugation of the label with the antibody can beachieved.

In certain embodiments, the detection method is a competitive bindingassay in which a competing anti-moesin antibody is used. Such competingantibody is capable of competing with moesin auto-antibodies for bindingto the peptides of the present application. In a competitive bindingassay, the reduction of binding signals can be indicative of theexistence and titer of the corresponding auto-antibodies.

Diagnostic Kits

For use in the applications described or suggested above, kits orarticles of manufacture are also provided by the present application.Such kits may comprise a carrier means being compartmentalized toreceive in close confinement one or more container means such as vials,tubes, and the like, each of the container means comprising one of theseparate elements to be used in the method. For example, one of thecontainer means may comprise a probe that is or can be detectablylabeled. Such probe may be a moesin fragment specific for anti-moesinautoantibody.

The kits of the present application will typically comprise thecontainer described above and one or more other containers comprisingmaterials desirable from a commercial and user standpoint, includingbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use. A label may be present on the container toindicate that the composition is used for a specific therapy ornon-therapeutic application, and may also indicate directions for eitherin vivo or in vitro use, such as those described above.

The kits of the present application have a number of embodiments. Atypical embodiment is a kit comprising a container, a label on saidcontainer, and a composition contained within said container; whereinthe composition includes a peptide of the present application that canbind to an anti-moesin autoantibody, the label on said containerindicates that the composition can be used to evaluate the presence ofanti-moesin autoantibodies in a sample, and instructions for using thepeptide of the present application for evaluating the presence ofanti-moesin autoantibodies in a sample. The kit can further comprise aset of instructions and materials for preparing a sample and applyingthe peptide of the present application to the sample. The kit mayinclude a secondary antibody, wherein the secondary antibody isconjugated to a label, e.g., an enzymatic label.

Other optional components in the kit include one or more buffers (e.g.,block buffer, wash buffer, substrate buffer, etc), other reagents suchas substrate (e.g., chromogen) which is chemically altered by anenzymatic label, epitope retrieval solution, control samples (positiveand/or negative controls), control slide(s) etc.

The following are examples of the methods and compositions of thepresent application. It is understood that various other embodiments maybe practiced, given the general description provided above.

EXAMPLES Example 1 Generation of Moesin Fragment Series

The following five moesin fragments are produced:

-   a. Moesin-1, containing amino acids 1-297 of human moesin protein    (SEQ ID NO:2), near N-terminal domain of the human moesin protein;-   b. Moesin-2, containing amino acids 298-577 of human moesin protein    (SEQ ID NO:3), near the helical and C-terminal tail domains of the    human moesin protein;-   c. Moesin-3, containing amino acids 298-470 of human moesin protein    (SEQ ID NO:4), near the helical domain of the human moesin protein;-   d. Moesin-4, containing amino acids 471-577 of human moesin protein    (SEQ ID NO:5), near the C-terminal tail domain of the human moesin    protein; and-   e. Moesin-5: full length human moesin protein, amino acid 1-577 (SEQ    ID NO:1).

The full length Moesin cDNA sequence (1-1734 bp) was obtained fromGenBank (GenBank NO: AB527296.1) and shown in FIG. 3. To generate theabove moesin fragments, PCR was used to amplify cDNA fragmentscorresponding to different amino acid fragments as described above.

PCR-amplified moesin DNA fragments were cloned into expression vectorsselected from pET32a(+) and pET28a(+). The constructed vectors were thenused to transform E. coli host cell line BL21(DE3) for culturing andexpression. The restriction and cloning maps of pET32a(+) and pET28a(+)are shown in FIGS. 4 and 5, respectively. The constructed expressionsystems for various moesin fragments were verified with restrictionenzyme digestion followed by sequencing to confirm the correct readingframe for expression of moesin fragments.

After sufficient culturing, host cells with expressed moesin fragmentswere harvested for collection and purification of moesin fragmentsaccording to standard protein expression protocols. The resultingprotein fragments were assayed with SDS-PAGE to confirm their identityand purity.

Example 2 Detection and Measurement of Specific Anti-MoesinAutoantibodies in Sera of Immune Thrombocytopenia Patients

Sera or plasma samples were collected from patients with various stagesof immune thrombocytopenia and tested for the presence of anti-moesinautoantibodies that recognize and bind to specific regions of the moesinprotein. Patients' profiles and clinical information were used tocategorize them based on types and stages of their diseases.

Moesin fragments obtained from Example 1 were used as antigens in ELISAassays for anti-moesin antibodies. Specifically, each micro well of theELISA plate was coated with about 400 ng of moesin fragment at 2° C. to8° C. for 12-16 hours, and then washed with PBS once before beingblocked with blocking solution and vacuum dried for storage and lateruse. So a highly purified Moesin fragment antigen was bound to the wellsof a polystyrene microwell plate under conditions that would preservethe antigen in its native state.

Sera samples were collected from two patient groups, i.e. immunethrombocytopenia and non-immune thrombocytopenia. The patient group ofimmune thrombocytopenia, from which the sera samples are collected fortesting consequently, had a total of 77 patients, which included 25patients that were clinically diagnosed as idiopathic thrombocytopenicpurpura, 17 patients that were clinically diagnosed as immune mediatedthrombocytopenia induced by drug therapy or blood transfusion, 35patients that were clinically diagnosed as thrombocytopenia accompanyingiron deficiency anemia. The patient group of non-immunethrombocytopenia, from which the sera samples were collected for testingconsequently, had a total of 47 patients, which included 9 patients thatwere clinically diagnosed as non-immune mediated thrombocytopenicpurpura of unknown cause, 11 patients that were clinically diagnosed asanaphylactoid purpura, 12 patients that were clinically diagnosed asmyelodysplastic syndrome, 15 patients that were clinically diagnosed asmultiple myeloma. Sera samples were also collected from 50 healthyindividuals and tested as healthy controls.

The controls and patient sera were diluted using PBS-T buffer (i.e. PBSbuffer containing 0.05% (v/v) of Tween-20), and 100 μl of such dilutedcontrols and diluted patient sera were then added to separate wells,allowing anti-moesin antibodies present to bind to the immobilizedantigen. Unbound sample was washed away using PBS-T buffer and an enzymelabeled anti-human IgG conjugate was added to each well. A secondincubation allowed the enzyme labeled anti-human IgG to bind to anyanti-moesin antibodies which have become attached to the micro wells.After washing away any unbound enzyme labeled anti-human IgG, theremaining enzyme activity was measured by adding a chromogenic substrate(H₂O₂/TMB) and measuring the intensity of the color that develops. 100μl of HRP Stop Solution (e.g. 2M H₂SO₄) were then added to each well.Sequence and timing of adding and maintaining HRP Stop Solution wereaccording to TMB Chromogen. Each ELISA plate was gently tapped withfingers to thoroughly mix the wells.

The assay was evaluated using a spectrophotometer to measure and comparethe color intensity that developed in the patient wells with the colorin the control wells. Specifically, bichromatic measurements are used tomeasure and compare the color intensity, wherein both OD₄₅₀ value andOD₆₃₀ value (as a reference) of each well were read within 15 mins ofstopping the reaction. The OD value of each test or control sample wascalculated by subtracting the OD₄₅₀ value with the OD₆₃₀ value.

The ELISA low positive control, the ELISA high positive control and theELISA negative control were run with every batch of samples to ensurethat all reagents and procedures performed properly. The ELISA negativecontrol was sera collected from healthy individuals. The OD values ofsera collected from 50 healthy individuals were each measured and theaverage OD value (the “Control OD Value”) and the standard deviation(the “Control Standard Deviation”) from those 50 samples werecalculated. Such Control OD Value and Control Standard Deviation wereused to determine the concentrations of the ELISA low positive controland high positive control. The ELISA low positive control contains serafrom patients with immune thrombocytopenia that were diluted enough toshow an OD value which equals to the Control OD Value plus three timesof the Control Standard Deviation. The ELISA high positive controlcontains sera from patients with immune thrombocytopenia that wasdiluted to show an OD value which equals to three times of the OD valueof the ELISA low positive control. The dilution was done using 0.01MPBS-T buffer.

The average OD value for each set of duplicates of a sample was firstdetermined, and the sample was determined positive if its average ODvalue was higher than the average OD value of the ELISA low positivecontrol (as shown in Table 1). The mean titer for each sample wasmeasured as the average OD value of the sample (as shown in FIG. 6).

As the skilled artisan will appreciate, the step of correlating a markerlevel to the presence or absence of immune thrombocytopenia can beperformed and achieved in different ways. In general a referencepopulation is selected and a normal range established. It is fairlyroutine to establish the normal range for anti-moesin antibodies usingan appropriate reference population. It is generally accepted that thenormal range depends, to a certain but limited extent, on the referencepopulation in which it is established. In one aspect, the referencepopulation is high in number, e.g., hundreds to thousands, and matchedfor age, gender and optionally other variables of interest. The normalrange in terms of absolute values, like a concentration given, alsodepends on the assay employed and the standardization used in producingthe assay.

The levels for anti-moesin antibodies can be measured and establishedwith the assay procedures given in the examples section. It has to beunderstood that different assays may lead to different cut-off values.

The clinical performance of a laboratory test depends on its diagnosticaccuracy, or the ability to correctly classify subjects into clinicallyrelevant subgroups. Diagnostic accuracy measures the test's ability tocorrectly distinguish different conditions of the subjects investigated.Such conditions are for example health and disease or benign versusmalignant disease.

That is, a significant higher value obtained from certain patientpopulation indicates the positive presence of the correspondinganti-moesin autoantibody.

The results of the experiments are listed in the following tablecomparing various patient groups for the positive presences of differentanti-moesin antibodies specific to certain moesin fragments (Table 1):

TABLE 1 Comparison of the Positive Presence of Anti-moesin Autoantibodyto Specific Moesin Fragments in Sera of Patient Groups and Control GroupNumber Anti- Each Moesin Fragment Positive of Moesin- Moesin- MoesinMoesin- Moesin- Patient Group Patient Subgroup Patients 1 3 4 2 5 ImmuneIdiopathic 25 22 0 (0) 18 19 21 Thrombocytopenia Thrombocytopenic(88.0%) (72.0%) (76.0%) (84.0%) Purpura Immune Mediated 17 17 1 13 15 16Thrombocytopenia  (100%) (5.9%) (76.5%) (88.2%) (94.1%) induced by drugtherapy, genetic factor or blood transfusion Thrombocytopenia 35 31 0(0) 25 24 32 Accompanying Iron (88.6%) (71.4%) (68.6%) (91.4%)Deficiency Anemia In total 77 70 1 56 58 69 (91.0%) (1.3%) (72.7%)(75.3%) (89.6%) Non-immune Non-immune 9 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)Thrombocytopenia Mediated Thrombocytopenic Purpura of unknown CauseAnaphylactoid Purpura 11 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Myelodysplastic12 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Syndrome Multiple Myeloma 15 0 (0) 0(0) 0 (0) 0 (0) 0 (0) In total 47 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) ControlHealthy Individuals 50 1 0 (0) 1 0 (0) 1  (2.0%)  (2.0%)  (2.0%)

As shown in Table 1, the positive presence of anti-moesin autoantibodiesthat specifically recognize and bind to the Moesin-1 (N-terminal FERMdomain), Moesin-4 (C-terminal tail domain), Moesin-2 (the fragmentcomprising the amino acids of helical and C-terminal tail domains) andMoesin-5 (the full length human moesin protein) significantly correlatedwith the incidence of immune thrombocytopenia.

The mean titers and standard deviations of anti-moesin autoantibodies tospecific moesin fragments in sera of patients with immunethrombocytopenia, non-immune thrombocytopenia and the control group ofhealthy individuals are listed in Table 2 below. The data of mean titersin Table 2 is also illustrated in FIG. 6, which shows that the amountsof the anti-moesin autoantibodies binding to moesin-1 and moesin-4 inthe sera of patients with immune thrombcytopenia are significantlyhigher than those in the sera of patient with non-immunethrombocytopenia and the healthy control group. Furthermore, in the seraof patients with immune thrombocytopenia, the amounts of the anti-moesinautoantibodies binding to moesin-1 and moesin-4 are significantly higherthan those binding to moesin-2, moesin-3 and moesin-5. The resultsindicate that moesin-1 (the N-terminal FERM domain) and moesin-4 (theC-terminal tail domain) can be used as indicators for diagnosis orprognosis of patients having or suspected of having immunethrombocytopenia.

TABLE 2 Mean Titers and Standard Deviations of Anti-MoesinAutoantibodies to Specific Moesin Fragments in Sera of Patient Groupsand Control Group Moesin Fragments Number of Mean Moesin- Group PatientsTiter/SD Moesin 1 Moesin 3 Moesin 4 Moesin 2 5 Immune 77 Mean Titer0.53163 0.259917 0.505167 0.229182 0.24925 Thrombocytopenia SD 0.3732490.082111 0.259787 0.041978 0.116492 Non-immune 47 Mean Titer 0.1863330.0965 0.110833 0.117333 0.105667 Thrombocytopenia SD 0.030164 0.0294130.024999 0.028724 0.028395 Control 50 Mean Titer 0.15525 0.1595 0.1440.099 0.0875 SD 0.018572 0.061701 0.037372 0.009592 0.009678

1. A method for diagnosing immune thrombocytopenia comprising (i)contacting a sample from a subject suspected of immune thrombocytopeniawith a first peptide capable of binding to an anti-moesin autoantibody,wherein the first peptide comprises a first moesin fragment consistingessentially of the C-terminal tail domain of human moesin protein or afragment thereof; (ii) detecting the binding of said first peptide to ananti-moesin autoantibody.
 2. The method of claim 1, wherein the firstpeptide comprises at least eight consecutive amino acid residues of theC-terminal tail domain of human moesin protein.
 3. The method of claim2, wherein the C-terminal tail domain of human moesin protein containsamino acid residues selected from the group consisting of amino acidresidues from the region between amino acid residues 471-487, 488-501,502-577, and 471-577 of human moesin protein.
 4. The method of claim 2,wherein the first peptide consists essentially of amino acid residues ofthe C-terminal tail domain of human moesin protein or a fragmentthereof.
 5. The method of claim 1, wherein the first peptide furthercomprises a carrier polypeptide.
 6. The method of claim 5, wherein thefirst peptide does not contain any substantial portion of the N-terminalFERM domain of human moesin protein.
 7. The method of claim 1, furthercomprising contacting the sample with a second peptide capable ofbinding to an anti-moesin autoantibody, wherein the second peptidecomprises a second moesin fragment consisting essentially of theN-terminal FERM domain of human moesin protein or a fragment thereof;and detecting the binding of said second peptide to an anti-moesinautoantibody.
 8. The method of claim 7, wherein the second peptidecomprises at least eight consecutive amino acid residues of theN-terminal FERM domain of human moesin protein.
 9. The method of claim8, wherein the N-terminal FERM domain of human moesin protein containsamino acid residues selected from the group consisting of amino acidresidues from the region between amino acid residues 1-94, 95-201,202-297, and 1-297 of human moesin protein.
 10. The method of claim 7,wherein the second peptide consists essentially of amino acid residuesof the N-terminal FERM domain of human moesin protein or a fragmentthereof.
 11. The method of claim 1, wherein the immune thrombocytopeniais primary immune thrombocytopenia.
 12. The method of claim 1, whereinthe sample is pre-tested with a blood counting method.
 13. The method ofclaim 1, wherein the binding of said first peptide to the anti-moesinautoantibody is detected through ELISA or immunoblotting. 14-17.(canceled)
 18. A kit for diagnosing immune thrombocytopenia, comprisinga) a peptide capable of binding to an anti-moesin autoantibody, whereinthe peptide comprises a moesin fragment consisting essentially of theC-terminal tail domain of human moesin protein or a fragment thereof;and b) a detecting reagent.
 19. The kit of claim 18, further comprisinga solid phase, wherein the peptide is bound to the solid phase.
 20. Themethod of claim 1 further comprising the following step: (iii)determining the pathological state of the subject according to acomparison of the level of the anti-moesin autoantibody obtained fromstep (ii) to a reference database obtained from diseased referencesamples correlating titers of the anti-moesin autoantibody topathological states of the immune thrombocytopenia.
 21. The method ofclaim 20, wherein the subject is undergoing a treatment for immunethrombocytopenia, and a decrease in the titer of the anti-moesinautoantibody is indicative of positive response of the subject to thetreatment.
 22. The kit of claim 18, wherein the peptide comprises atleast eight consecutive amino acid residues of the C-terminal taildomain of human moesin protein.
 23. The kit of claim 22, wherein theC-terminal tail domain of human moesin protein contains amino acidresidues selected from the group consisting of amino acid residues fromthe region between amino acid residues 471-487, 488-501, 502-577, and471-577 of human moesin protein.
 24. The kit of claim 22, wherein thepeptide consists essentially of amino acid residues of the C-terminaltail domain of human moesin protein or a fragment thereof.